Converting parathyroid hormone-related peptide (PTHrP) into a potent PTH-2 receptor agonist

Most of the bone and kidney-related functions of parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) are thought to be mediated by the PTH/PTHrP receptor. Recently, a homologous receptor, the PTH-2 receptor, was obtained from rat and human brain cDNA libraries. This receptor displayed the remarkable property of responding potently to PTH, but not to PTHrP. To begin to define residues involved in the ligand specificity of the PTH-2 receptor, we studied the interaction of several PTH/PTHrP hybrid ligands and other related peptide analogs with the human PTH-2 receptor. The results showed that two sites in PTH and PTHrP fully account for the different potencies that the two ligands exhibited with PTH-2 receptors; residue 5 (His in PTHrP and Ile in PTH) determined signaling capability, while residue 23 (Phe in PTHrP and Trp in PTH) determined binding affinity. By changing these two residues of PTHrP to the corresponding residues of PTH, we were able to convert PTHrP into a ligand that avidly bound to the PTH-2 receptor and fully and potently stimulated cAMP formation. Changing residue 23 alone yielded [Trp23]hPTHrP-(1-36), which was an antagonist for the PTH-2 receptor, but a full agonist for the PTH/PTHrP receptor. Residues 5 and 23 in PTH and PTHrP thus play key roles in signaling and binding interactions, respectively, with the PTH-2 receptor. Receptor-selective agonists and antagonists derived from these studies could help to identify the biological role of the PTH-2 receptor and to map specific sites of ligand-receptor interaction.     

Cell-specific signal transduction of parathyroid hormone (PTH)-related protein through stably expressed recombinant PTH/PTHrP receptors in vascular smooth muscle cells

PTH-related protein activates a G protein-coupled PTH/PTHrP receptor in many cell types and produces diverse biological actions. To study the signal transduction events associated with biological activity of the PTH/PTHrP receptor in vascular smooth muscle, a principal PTHrP-responsive tissue, rat aortic smooth muscle cells (A10) were stably transfected with a plasmid encoding a PTH/PTHrP receptor and tested for ligand binding, PTHrP-(1-34)-induced cAMP levels, inositol phosphate production, and cytosolic calcium transients. Of nineteen G418-resistant lines recovered, all exhibited high affinity binding [approximately dissociation constant (Kd) > 10(-10)) of iodinated [Tyr36]hPTHrP(1-36)NH2 and ligand-induced cAMP accumulation (2- to 100-fold), which was directly proportional to PTH/PTHrP receptor number (range 4 x 10(3) to 7 x 10(7) sites/cell]. PTHrP had no effect on intracellular calcium or inositol phosphate formation in any cell line regardless of receptor number despite the presence of detectable G alpha q). Transient overexpression of individual G alpha q proteins (G alpha q, G alpha 11 or G alpha 14) into PTH/PTHrP receptor-expressing A10 cells conferred the ability of PTHrP to increase intracellular calcium and inositol phosphate formation. Ligand activation of the recombinant PTH/PTHrP receptor elicited appropriate downstream biological effects in A10 cells including inhibition of DNA synthesis and osteopontin messenger RNA (mRNA) expression. Thus, a single PTH/PTHrP receptor, though capable of coupling to different G proteins, signals exclusively through a cAMP-dependent pathway in vascular smooth muscle.     

Transmembrane residues of the parathyroid hormone (PTH)/PTH-related peptide receptor that specifically affect binding and signaling by agonist ligands

Polar residues within the transmembrane domains (TMs) of G protein-coupled receptors have been implicated to be important determinants of receptor function. We have identified mutations at two polar sites in the TM regions of the rat parathyroid hormone (PTH)/PTH-related peptide receptor, Arg-233 in TM 2 and Gln-451 in TM 7, that caused 17-200-fold reductions in the binding affinity of the agonist peptide PTH-(1-34) without affecting the binding affinity of the antagonist/partial agonist PTH-(3-34). When mutations at the TM 2 and TM 7 sites were combined, binding affinity for PTH-(1-34) was restored to nearly that of the wild type receptor. The double mutant receptors, however, were completely defective in signaling cAMP or inositol phosphate production in response to PTH-(1-34) agonist ligand. The results demonstrate that Arg-233 and Gln-451 have important roles in determining agonist binding affinity and transmembrane signaling. Furthermore, the finding that residues in TM 2 and TM 7 are functionally linked suggests that the TM domain topology of the PTH/PTH-related peptide receptor may resemble that of receptors in the rhodopsin/beta-adrenergic receptor family, for which structural and mutagenesis data suggest interactions between TMs 2 and 7.     

Effects of parathyroid hormone (PTH)-related protein and PTH on osteoclasts and osteoclast precursors in vivo

Increased production of PTH-related protein (PTHrP) and PTH is frequently responsible for hypercalcemia and its associated morbidity. However, it is unclear whether these peptides produce identical effects on cells in the osteoclast lineage in vivo. To examine the effects of continuous in vivo exposure to these factors on both the osteoclast precursors and mature osteoclasts, we inoculated Chinese hamster ovarian cells expressing PTH-(1-84), PTHrP-(1-141), or nontransfected Chinese hamster ovarian cells into nude mice. The effects of these tumors on blood ionized calcium, plasma PTH and PTHrP concentrations, and osteoclast formation were then determined. PTH and PTHrP tumor-bearing mice became hypercalcemic (1.90 +/- 0.04 and 1.97 +/- 0.16 mmol/liter, respectively) compared with control mice (1.29 +/- 0.015 mmol/liter). After 4 days of hypercalcemia, mice were killed, and bone marrow cells were harvested to examine cells at three discrete stages of osteoclast development: multipotent osteoclast precursors, the granulocyte/macrophage colony-forming unit; more committed marrow mononuclear osteoclast precursors; and mature osteoclasts. Neither PTH nor PTHrP had an effect on granulocyte/macrophage colony-forming unit, but similarly increased the number of more committed mononuclear osteoclast progenitors as well as mature osteoclasts in the calvaria. No differences were detected between the effects of PTH and PTHrP on cells in the osteoclast lineage in vivo. Thus, PTH and PTHrP appear to affect only more differentiated cells in the osteoclast lineage, and the differences in osteoclastic bone resorption between primary hyperparathyroidism and humoral hypercalcemia of malignancy are probably due to mechanisms other than effects on osteoclast precursor cells in vivo.     

Parathyroid hormone-related protein-(1-36) is biologically active when administered subcutaneously to humans

PTH-related protein (PTHrP) is responsible for most cases of humoral hypercalcemia of malignancy (HHM). It mimics the actions of PTH as a result of its structural homology with PTH and its ability to bind to and signal via the PTH/PTHrP receptor in bone and kidney. PTHrP-(1-36) appears to be one of several secretory forms of PTHrP. This peptide has been administered iv to normal volunteers previously and has been shown to produce effects that are qualitatively and quantitatively the same as those produced by PTH-(1-34). To determine whether PTHrP-(1-36) could be used sc in humans as a diagnostic reagent for elucidating the differences between HHM and hyperparathyroidism, we performed a 12-h dose-finding study examining whether sc PTHrP-(1-36) could elicit effects on mineral homeostasis. PTHrP-(1-36) administered sc in three doses (0.82, 1.64, and 3.28 micrograms/kg) to 21 normal women produced increases in circulating PTHrP-(1-36), reductions in serum phosphorus and the renal phosphorus threshold, increments in fractional calcium excretion and nephrogenous cAMP excretion, and increases in plasma 1,25-dihydroxyvitamin D. These changes were highly significant in statistical terms and were observed at doses that had no effect on serum calcium or endogenous PTH. These studies demonstrate the feasibility of using PTHrP-(1-36) as a diagnostic probe for future studies aimed at elucidating the differing pathophysiologies of HHM and hyperparathyroidism.     

Mono- and bicyclic analogs of parathyroid hormone-related protein. 1. Synthesis and biological studies

The bioactive conformation of parathyroid hormone-related protein (PTHrP), a single-chain linear peptide structurally similar to parathyroid hormone (PTH), is of considerable interest because PTH and PTHrP both recognize and bind to a shared G-protein-coupled receptor. Both hormones are thought to present a bioactive conformation to the receptor which is substantially alpha-helical in nature. To better characterize this putative biologically relevant conformation, we prepared a series of conformationally constrained analogs of PTHrP with enhanced alpha-helical stability. A combination of structural constraint and helix stabilization was achieved through side chain-to-side chain lactam ring formation between Lys(i) and Asp(i+4) residues (13-to-17 and 26-to-30) along the PTHrP sequence. Mono- and bicyclic analogs derived from the agonist PTHrP-(1-34)NH2 and the antagonist PTHrP-(7-34)NH2 were prepared and characterized in terms of receptor binding and stimulation (or antagonism) of PTH-stimulated adenylyl cyclase activity in osteoblast-like cells. The binding affinity of monocyclic [Lys13,Asp17]-(I) and bicyclic [Lys13,Asp17,Lys26,Asp30]PTHrP-(1-34)NH2 (III) agonists was in the low nanomolar range and similar to that of the parent linear peptide. Furthermore, their efficacy was in the sub-nanomolar range and about 10-fold higher than that of the corresponding linear parent peptide. Analogs I and III are the first cyclic PTH/PTHrP receptor agonists and amongst the most potent PTHrP analogs yet designed. The rank-order of potency in the cyclic antagonist series does not correlate with the binding affinities. In light of the positional dependence and the differential effects of lactam bridge formation on the biological activities of agonist vs antagonists, these analogs may provide insight regarding the biologically relevant conformations of PTHrP-derived ligands [Maretto et al. (1997) Biochemistry 36, 3300-3307].     

Targeted expression of constitutively active receptors for parathyroid hormone and parathyroid hormone-related peptide delays endochondral bone formation and rescues mice that lack parathyroid hormone-related peptide

Mice in which the genes encoding the parathyroid hormone (PTH)-related peptide (PTHrP) or the PTH/PTHrP receptor have been ablated by homologous recombination show skeletal dysplasia due to accelerated endochondral bone formation, and die at birth or in utero, respectively. Skeletal abnormalities due to decelerated chondrocyte maturation are observed in transgenic mice where PTHrP expression is targeted to the growth plate, and in patients with Jansen metaphyseal chondrodysplasia, a rare genetic disorder caused by constitutively active PTH/PTHrP receptors. These and other findings thus indicate that PTHrP and its receptor are essential for chondrocyte differentiation. To further explore the role of the PTH/PTHrP receptor in this process, we generated transgenic mice in which expression of a constitutively active receptor, HKrk-H223R, was targeted to the growth plate by the rat alpha1 (II) collagen promoter. Two major goals were pursued: (i) to investigate how constitutively active PTH/PTHrP receptors affect the program of chondrocyte maturation; and (ii) to determine whether expression of the mutant receptor would correct the severe growth plate abnormalities of PTHrP-ablated mice (PTHrP-/-). The targeted expression of constitutively active PTH/PTHrP receptors led to delayed mineralization, decelerated conversion of proliferative chondrocytes into hypertrophic cells in skeletal segments that are formed by the endochondral process, and prolonged presence of hypertrophic chondrocytes with delay of vascular invasion. Furthermore, it corrected at birth the growth plate abnormalities of PTHrP-/- mice and allowed their prolonged survival. "Rescued" animals lacked tooth eruption and showed premature epiphyseal closure, indicating that both processes involve PTHrP. These findings suggest that rescued PTHrP-/- mice may gain considerable importance for studying the diverse, possibly tissue-specific role(s) of PTHrP in postnatal development.     

The parathyroid hormone (PTH)/PTH-related peptide receptor mediates actions of both ligands in murine bone

PTH and PTH-related peptide (PTHrP) have been shown to bind to and activate the same PTH/PTHrP receptor. Recent studies have demonstrated, however, the presence of additional receptors specific for each ligand. We used the PTHrP and PTH/PTHrP receptor gene knock-out models to investigate whether this receptor mediates the actions of both ligands in bone. The similar phenotype of the PTHrP (-/-) and PTH/PTHrP receptor (-/-) animals in the growth plate of the tibia suggests that this receptor mediates the actions of PTHrP. Electron microscopic studies have confirmed the accelerated differentiation and disordered organization of chondrocytes, with the accumulation of large amounts of dispersed glycogen granules in the cytoplasm of proliferative and maturing cells of both genotypes. The contrasting growth plate mineralization patterns of the PTHrP (-/-) and PTH/PTHrP receptor (-/-) mice, however, suggest that the actions of PTHrP and the PTH/PTHrP receptor are not identical. Studies using calvariae from PTH/PTHrP receptor (-/-) embryos demonstrate that this receptor solely mediates the ability of PTH and PTHrP to stimulate adenylate cyclase in bone and to stimulate bone resorption. Furthermore, we show that osteoblasts of PTH/PTHrP receptor (-/-) animals, but not PTHrP (-/-) animals, have decreased levels of collagenase 3, osteopontin, and osteocalcin messenger RNAs. The PTH/PTHrP receptor, therefore, mediates distinct physiologic actions of both PTH and PTHrP.     

Parathyroid hormone (PTH) stimulates adrenocorticotropin release in AtT-20 cells stably expressing a common receptor for PTH and PTH-related peptide

Complementary DNA encoding a rat bone PTH/PTHrP receptor was stably expressed in the murine corticotroph cell line, AtT-20. Several clones, expressing variable numbers of PTH/PTHrP receptors, were developed. In contrast to the relatively low binding affinity (apparent Kd = 15 nM) observed in COS-7 cells transiently expressing the PTH/PTHrP receptor, all AtT-20 stable transfectants bound [Nle8,18,Tyr34]bPTH(1-34)NH2 (NlePTH) with an affinity that was indistinguishable from that observed in ROS 17/2.8 cells expressing native PTH/PTHrP receptors. Additionally, NlePTH dramatically increased cAMP accumulation and ACTH release in AtT-20 cells expressing the PTH/PTHrP receptor with an ED50 of 0.6 +/- 0.3 and 0.3 +/- 0.1 nM, respectively. The high binding affinity and the high efficacy of NlePTH in stimulating cAMP accumulation and ACTH release indicate that the PTH/PTHrP receptor is efficiently coupled to the intracellular signalling system responsible for stimulation of ACTH release in AtT-20 cells. No additivity of cAMP accumulation or of ACTH release was observed when these cells were treated with maximally active concentrations of both NlePTH and CRF. This suggests that the receptors for both of these hormones share the same intracellular effectors, and that intracellular signaling in AtT-20 cells is not compartmentalized. Additionally, the ability of NlePTH to stimulate ACTH release in AtT-20 cells, a function that is normally performed by CRF, demonstrates promiscuity between activated receptors and distal biological functions.     

Role of glycosylation in expression and function of the human parathyroid hormone/parathyroid hormone-related protein receptor

Parathyroid hormone (PTH) regulates calcium metabolism through a specific G protein-coupled, seven-transmembrane helix-containing receptor. This receptor also binds and is activated by PTH-related protein (PTHrP). The human (h) PTH/PTHrP receptor is a membrane glycoprotein with an apparent molecular weight of approximately 85000 which contains four putative N-glycosylation sites. To elucidate the functional role of receptor glycosylation, if any, we studied hormone binding and signal transduction in human embryonic kidney cells transfected with hPTH/PTHrP receptor (HEK-293/C-21). These cells stably express 300000-400000 receptors per cell. Inhibition of N-glycosylation with an optimized concentration of tunicamycin yielded completely nonglycosylated hPTH/PTHrP receptor (approximately 60 kDa). This receptor form is fully functional; it maintains nanomolar binding affinity for PTH- and PTHrP-derived agonists and antagonists. PTH and PTHrP agonists stimulate cyclic AMP accumulation and increases in cytosolic calcium levels. In addition, the highly potent benzophenone (pBz2)-containing PTH-derived radioligand [Nle8,18,Lys13(epsilon-pBz2),L-2-Nal23,Tyr34 3-125I)]bPTH(1-34)NH2 can photoaffinity cross-link specifically to the nonglycosylated receptor. The molecular weight (approximately 60000) of the band representing the photo-cross-linked, nonglycosylated receptor (obtained from the tunicamycin-treated HEK-293/C-21 cells) was similar to that of the deglycosylated photo-cross-linked receptor (obtained by enzymatic treatment with Endoglycosidase-F/N-glycosidase-F). Our findings indicate that glycosylation of the hPTH/PTHrP receptor is not essential for its effective expression on the plasma membrane or for the binding of ligands known to interact with the native receptor. The nonglycosylated hPTH/PTHrP receptor remains fully functional with regard to both of its known signal transduction pathways: cAMP-protein kinase A and phospholipase C-cytosolic calcium.     

Centrally administered parathyroid hormone (PTH)-related protein(1-34) but not PTH(1-34) stimulates arginine-vasopressin secretion and its messenger ribonucleic acid expression in supraoptic nucleus of the conscious rats

It has been suggested that PTH-related protein (PTHrP) is an endogenous modulator of cardiovascular systems. We have reported that PTHrP(1-34), but not PTH(1-34), causes the release of arginine-vasopressin (AVP) from the supraoptic nucleus (SON) of the hypothalamus in vitro through a novel receptor distinct from the PTH/PTHrP receptors (type I or type II) described previously. In this study, we have investigated the in vivo effects of PTHrP(1-34) on AVP secretion and its, messenger RNA (mRNA) expression in the SON in conscious rats. Intracerebroventricular (i.c.v.) administration of PTHrP(1-34) resulted in an increase in plasma AVP concentration in a dose-dependent manner (0-400 pmol/rat). The maximal effect was obtained at 15 min after i.c.v. administration of PTHrP(1-34). Neither PTHrP(7-34) nor PTH(1-34) had any effect on plasma AVP levels. PTHrP(1-34)-induced AVP secretion was antagonized by pretreatment with PTHrP(7-34) but not by that with PTH(1-34). In addition, in situ hybridization study revealed that AVP mRNA expression in the SON and paraventricular nucleus was significantly increased 30 min after i.c.v. administration of PTHrP(1-34) and reached a maximum at 180 min. Furthermore, in Northern blot analyses, AVP mRNA expression in the SON was increased to approximately a 2-fold of basal level by PTHrP(1-34). On the other hand, neither PTHrP(7-34) or PTH(1-34) had any effect on the mRNA expression. The PTHrP(1-34)-stimulated AVP mRNA expression was eliminated by pretreatment with PTHrP(7-34) but not with PTH(1-34). These results suggest that, in the central nervous system, PTHrP(1-34) is involved in AVP secretion through a novel receptor distinct from the PTH/PTHrP receptors reported previously, playing a role in the body water and electrolyte homeostasis.     

A homozygous inactivating mutation in the parathyroid hormone/parathyroid hormone-related peptide receptor causing Blomstrand chondrodysplasia

We describe a patient with Blomstrand chondrodysplasia, a lethal genetic disorder characterized by extremely advanced endochondral bone maturation, in whom a homozygous missense mutation is present in the gene coding for the PTH/PTHrP receptor that leads to the substitution of a proline for a leucine in the N-terminal portion of the receptor (P132L). PTH-induced cAMP accumulation was severely reduced in COS-7 cells expressing P132L receptors compared to that of cells expressing wild-type receptors, and PTH-induced inositol phosphate accumulation was not detectable in cells expressing the mutant receptor. Similar results were obtained using PTHrP as an agonist. Maximal specific binding of radioiodinated [Tyr36]PTHrp(1-36) by cells transfected with the P132L receptor was < 10% of that observed for cells transfected with the wild-type receptor. Despite the reduction in radioligand binding to P132L receptors, the intensity and distribution of the fluorescent signal resulting from the expression of receptors fused to GFP were similar for cells transfected with the wild-type and mutant P132L receptors, suggesting a similar degree of cell surface expression. These results firmly establish the role of abnormalities in the PTH/PTHrP receptor in the pathogenesis of Blomstrand chondrodysplasia, and thereby confirm the importance of signaling through the PTH/PTHrP receptor in human fetal skeletal development. Because the amino-acid mutated in the patient described here is otherwise conserved in all mammalian class II G protein-coupled receptors, this abnormality may provide insights into structural features needed for the normal function of this family of receptors.     

Conditionally immortalized murine osteoblasts lacking the type 1 PTH/PTHrP receptor

Osteoblasts synthesize and mineralize bone matrix and are principal target cells for parathyroid hormone (PTH). The type 1 PTH/PTH-related protein (PTHrP) receptor (PTH1R), cloned from rat osteoblastic cells, activates multiple intracellular signaling mechanisms. The specific roles of these PTH1R signals, or of responses to other types of PTH receptors that may be expressed, in regulating osteoblast function are incompletely understood. Use of established mammalian osteoblastic cell lines has led to much understanding of PTH action in bone, although such cells are of neoplastic origin or have other characteristics that compromise their validity as models of normal osteoblasts. To examine the role of the PTH1R in osteoblast biology, we have isolated a series of clonal murine calvarial osteoblastic cell lines that are only conditionally immortalized, via expression of a transgene encoding the tsA58 temperature-sensitive SV40 large T antigen, and that lack both functional alleles of the PTH1R gene. When cultured under nontransforming conditions, these cells stopped proliferating, expressed a series of characteristic osteoblastic genes (including the nonfunctional remnant of the PTH1R gene), and, after 3-4 weeks, produced mineralized bone nodules in a manner that was regulated by 1,25-dihydroxyvitamin D3 but not by PTH(1-84). Cyclic AMP measurements revealed no evidence of expression of alternate species of Gs-linked PTH receptors. Stable transfection with PTH1R cDNA reconstituted both PTH binding and adenylyl cyclase activation, increased basal osteocalcin expression, and supported PTH stimulation of c-Fos expression and matrix mineralization. These conditionally transformed, PTH1R(-/-) clonal osteoblastic cell lines should prove useful for studies of the regulation of osteoblast differentiation and function by both endogenous nonclassical species of PTH (or PTHrP) receptors and mutant signal-selective PTH1Rs.     

Macrophage colony-stimulating factor release and receptor expression in bone cells

Colony-stimulating factors (CSF) may play a role in bone resorption. To examine whether osteoblasts secrete colony-stimulating activity (CSA) in response to parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), conditioned medium (CM) from ROS 17/2.8 cells and primary rat osteoblasts were examined for induction of clonal growth of cultured rat bone marrow cells. Untreated cells constitutively secreted CSA, which increased with PTH and PTHrP treatment. The colonies formed were principally comprised of macrophages, and preincubation of CM with antiserum to murine macrophage colony-stimulating factor (M-CSF) neutralized most of the CSA, suggesting that the osteoblast-derived CSA was predominantly due to M-CSF. PTHrP treatment upregulated steady-state M-CSF mRNA levels. To investigate a paracrine role for M-CSF in bone we examined bone tissue and cells for the M-CSF receptor c-fms using immunohistochemical techniques and demonstrated staining of mature osteoclasts both in situ and after isolation. We conclude that M-CSF is responsible for the majority of the CSA released by PTH- and PTHrP-treated rat osteoblasts. In addition we identified CSF-1 receptor expression in mature osteoclasts. These data suggest that M-CSF is a mediator of osteoblast-osteoclast interaction in PTH- and PTHrP-induced bone resorption.     

Identification of transmembrane domain residues determinant in the structure-function relationship of the human platelet-activating factor receptor by site-directed mutagenesis

Platelet-activating factor (PAF) is a potent phospholipid mediator that produces a wide range of biological responses. The PAF receptor is a member of the seven-transmembrane GTP-binding regulatory protein-coupled receptor superfamily. This receptor binds PAF with high affinity and couples to multiple signaling pathways, leading to physiological responses that can be inhibited by various structurally distinct PAF antagonists. We have used site-directed mutagenesis and functional expression studies to examine the role of the Phe97 and Phe98 residues located in the third transmembrane helix and Asn285 and Asp289 of the seventh transmembrane helix in ligand binding and activation of the human PAF receptor in transiently transfected COS-7 cells. The double mutant FFGG (Phe97 and Phe98 mutated into Gly residues) showed a 3-4-fold decrease in affinity for PAF, but not for the specific antagonist WEB2086, when compared with the wild-type (WT) receptor. The FFGG mutant receptor, however, displayed normal agonist activation, suggesting that these two adjacent Phe residues maintain the native PAF receptor conformation rather than interacting with the ligand. On the other hand, substitution of Ala for Asp289 increased the receptor affinity for PAF but abolished PAF-dependent inositol phosphate accumulation; it did not affect WEB2086 binding. Substitution of Asn for Asp289, however, resulted in a mutant receptor with normal binding and activation characteristics. When Asn285 was mutated to Ala, the resulting receptor was undistinguishable from the WT receptor. Surprisingly, substitution of Ile for Asn285 led to a loss of ligand binding despite normal cell surface expression levels of this mutant, as verified by flow cytometric analysis. Our data suggest that residues 285 and 289 are determinant in the structure and activation of the PAF receptor but not in direct ligand binding, as had been recently proposed in a PAF receptor molecular model.